Creatinine is a metabolic product produced by degradation of both creatine and phosphorcreatine. Creatinine is found in both the blood and urine. It is ultimately excreted in the urine at a relatively constant rate. Creatinine levels in the body fluids represent a highly useful indicator of renal disease and certain muscular diseases. Normal concentrations of creatinine in blood serum typically are within the range of from about 0.5 to about 1.7 mg per 100 ml of serum.
Creatinine iminohydrolase is an enzyme which specifically catalyzes the hydrolysis of creatinine to ammonia. Accordingly, by contacting an aqueous liquid containing creatinine with this enzyme to generate ammonia, the presence and/or concentration of creatinine in the liquid can be determined by detecting the level of generated ammonia. This enzyme can therefore play an important role in the clinical laboratory where it can be used as a diagnostic test reagent for the determination of creatinine in biological liquids.
Creatinine iminohydrolase, sometimes referred to as creatinine desimidase, has heretofore been obtained from various microorganisms. For example, J. Szulmajster, in J. Bacteriol, 75: 633, 1958 and Biochim Biophys Acta, 30: 154, 1958 describes a preparation of creatinine iminohydrolase from an anaerobic, gram-positive microorganism Clostridium paraputrificum. In addition, U.S. Pat. No. 4,087,329 and 4,134,793 describe the production of creatine iminohydrolase from several aerobic sources including microorganisms from the genera Brevibacterium, Corynebacterium, Pseudomonas and Arthrobacter. Also, Thompson and Rechnitz, Analytical Chemistry, Vol. 46, No. 2, February, 1974 at p. 246 describe a creatinine iminohydrolase enzyme obtained from Beckman Inc., Microbics Operations, LaHabra, California and the use of this enzyme to assay for creatinine.
In the production of creatinine iminohydrolase from a microorganism source, the desired enzyme is generally extracted from the microbial cells or the nutrient medium in which the cells are cultured together with various other enzymes capable of producing ammonia, particularly urease. Urease contamination of creatinine iminohydrolase enzyme preparations presents a serious problem because both urea, the substrate for urease, and creatinine, the substrate for creatinine iminohydrolase, are typically present in serum and other biological fluids. Moreover, urea is typically present in serum in much greater concentration than creatinine. Thus, an enzymatic assay of serum creatinine by use of a creatinine iminohydrolase enzyme preparation contaminated with urease leads to significant assay error through the production of spurious amounts of ammonia.
To overcome the urease contamination problem, creatinine iminohydrolase preparations must typically undergo extensive purification procedures to remove the urease contaminant. This is specifically noted, for example, at page 248 of the aforementioned Thompson and Rechnitz publication. In their publication, Thompson and Rechnitz recommend the use of ion exchange chromatography using DEAE cellulose beads (available from Pharmacia, Uppsala, Sweden) to purify the enzyme. Similarly, the aforementioned U.S. Pat. Nos. 4,087,329 and 4,134,793 also employ ion exchange chromatography using DEAE cellulose beads to remove protein contaminants, presumably enzyme contaminants, from the crude creatinine desimidase preparations described in these patents. Accordingly, the discovery of a urease-free creatinine iminohydrolase enzyme preparation would represent a particularly valuable contribution to the art.